Process for preparing a carrier useful in immunoassays by deposition of a complex of a specifically binding substance with hydrophobic protein, and the resulting carrier

Abstract

The present invention provides a process for the preparation of a specifically bindable protein substance bound to an insoluble carrier material, especially for use in a heterogeneous analysis process, wherein a soluable protein with a molecular weight above about 500,000, which is more hydrophobic than the specifically bindable substance, is coupled to the specifically bindable substance and then the conjugate of reaction component and protein is adsorbed on a hydrophobic solid phase. The present invention also provides a carrier material for use in solid phase immunoassays including a hydrophobic solid phase which is adsorbed on a conjugate of a hydrophobic protein with a molecular weight above about 500,000 and of a specifically bindable protein or protein containing substance.

We claim: 
     
       1. A process for preparing an insoluble carrier material useful in binding assays comprising; forming a cross-linked conjugate of a water-soluble first protein having a molecular weight of at least 500,000 daltons with a second protein substance which conjugates by cross-linking with the water-soluble first protein wherein the water-soluble first protein is more hydrophobic than said second protein substance,   adsorbing the cross-linked conjugate on a hydrophobic solid phase material to form said insoluble carrier material, wherein the second protein substance consists of one member of a specifically bindable pair of substances wherein the binding member is other than the water-soluble protein so that the cross-linked conjugate is specifically bindable.   
     
     
       2. The process according to claim 1, wherein said water-soluble protein has a molecular weight in the range of 500,000 to 20 million daltons. 
     
     
       3. The process according to claim 1, and further including the step of preparing said water-soluble protein prior to the forming of said cross-linked conjugate, the preparing of said water-soluble protein including linking a precursor protein having an original molecular weight in the range of 10,000 to 700,000 daltons with at least one other molecule to form a molecule having a molecular weight greater than the molecular weight of the precursor protein and in the range of 500,000 to 20 million daltons. 
     
     
       4. The process according to claim 3 and the linking of said precursor protein including cross-linking said precursor protein prior to conjugation with one or more reagents selected from the group consisting of bifunctional protein reagents and polyfunctional protein reagents. 
     
     
       5. The process according to claim 1 and further including the step of preparing said water-soluble protein from a precursor protein prior to forming the cross-linked conjugate, said preparing of said water-soluble protein including a step of causing the precursor protein to become more hydrophobic. 
     
     
       6. The process according to claim 5 and the step of causing the precursor protein to become more hydrophobic being accomplished by a method selected from the group consisting of heat treatment, treatment with acid, treatment with denaturing agents, treatment with chaotropic ions, and treatment to cause chemical coupling with a hydrophobic compound. 
     
     
       7. The process according to claim 4, and the cross-linking of the precursor protein including cross-linking with disuccinimidyl suberate. 
     
     
       8. The process according to claim 3, and said precursor protein being selected from the group consisting of bovine serum albumin, lipase, and immune γ-globulin. 
     
     
       9. The process of claim 1 wherein the binding assay is an immunoassay. 
     
     
       10. A specifically bindable complex adapted for adsorption onto a hydrophobic solid phase, said complex comprising: a water-soluble first protein coupled with a second protein substance to form a cross-linked conjugate therewith wherein said second protein is one of a binding pair of substances which specifically bind with each other and which conjugate is specifically bindable with the other substance of the binding pair, said soluble first protein having a molecular weight of at least about 500,000 daltons and being more hydrophobic than said second protein substance.   
     
     
       11. A carrier material comprising: a hydrophobic solid phase; and   a crook-linked conjugate being absorbedly supported on said solid phase said cross-linked conjugate comprising   a water-soluble hydrophobic first protein having a molecular weight of at least about 500,000 daltons and being coupled with a second protein substance to form said cross-linked conjugate, wherein the second protein is one of a binding pair of substances which specifically bind with each other and said cross-linked conjugate is specifically bindable with the other substance of the binding pair of substances.   
     
     
       12. The carrier material according to claim 11, wherein said hydrophobic solid phase is of a material selected from the group consisting of polystyrene, polymethacrylate, polyamide, polytetrafluoroethylene, and copolymers of styrene and acrylonitrile. 
     
     
       13. The carrier material according to claim 11, and said hydrophobic protein being derived from a precursor protein selected from the group consisting of bovine serum albumin, lipase, and immune ↓-globulin, which precursor protein is treated to increase the molecular weight and the hydrophobic quality of said precursor protein. 
     
     
       14. The carrier material of claim 11 wherein the second protein substance is selected from the group consisting of antigens, and fragments thereof. 
     
     
       15. The carrier material of claim 11, wherein the second protein substance is selected from the group consisting of streptavidin and avidin. 
     
     
       16. The carrier material of claim 11 wherein said solid phase is a fleece containing material selected from the group consisting of glass fibers, and   mixtures of polymer fibers and fibers of cellulose or cellulose ester.